| Objective Chronic myeloid leukemia (CML), with of chromosome, is one of primary clonal malignancies of hemapoietic stem cell. Philadelphia (Ph) chromosome is the hallmark of malignancies clone in CML.The Ph chromosome is a translocation of the ABL proto-oncogene from chromosome 22 to the long arm of chromosome 9, t(9; 22). The result of this translocation is the approximation of the ABL gene to the breakpoint cluster region (BCR) to form a bcr/abl fusion gene and expresses generally a P210 bcr/abl fusion protein. The p210bcr/abl fusion protein is considered as the mainstay pathophysiologic cause of CML. Because the p210bcr/abl fusion protein has stronger protein tyrosine kinase (PTK) activity than the c-abl protein, it can activates many signaling transmission paths and results in promoting the proliferation , inhibiting the apoptosis and decreaseing the adhesion properties of cells. Recently Some studies indicate that bcr gene might be the down-regulate gene of bcr/abl gene. When bcr gene expresses, the function of P210bcr/abl is SUppressed. .The phosphorylation of the 354th amino acid serine of bcr protein is the crucial step of the bcr suppression on bcr/abl process. Additionally, there is a SH2 binding site of GRB2 in bcr gene, the phosphorylation of tyr177 in this site can induce the phosphorylation of GRB2 and results in the activation of RAS paths and promote theproliferation of cells. To verify the down-regulation effect of bcr gene on bcr/abl gene, we reconstructed the shorter segmented bcr gene which includes 354th amino acid serine and transferred it to K562 cells, and observed it whether had the same function as that of omni-longus bcr gene. If the K562 cell line with target gene sustaining stable expression is obtained, it will make ours exploring bcr gene and the interaction between bcr and bcr/abl, as well as testing the hypothesis we have supposed: when bcr/abl gene express in some normal human hematopoitic stem cell, it induced the expression or higher expression of bcr. The expression or higher expression of bcr might induce the apoptosis of bcr/abl positive cells, which may explain the molecular mechanism of the evolution of CML and helps to found the basement for therapeutic molecular target.Methods Subclone the segmented bcr from normal human peripheral blood with PCR. Reconstruct PEGFP-Bcr system, the carrier of integrated expression of Bcr genetic green fluorescent protein in mammal cells. After successful reconstruction of the target gene expression vector, we transfered it to k562 cell line by liposome kit. The results and rates of transfection were observed with fluorescent microscope and flow cytometry at 48 hrs after transfection. The transfected K562 cell was selected by neomycin (G418). (K562 cell transfected by PEGFP-N1 named as K562control, K562 cell transferred by PEGFP-BCR named as K562eGFP-bcr). The protein expressed by target gene was identified by Western-blotting. The proliferative capacity ofK562control and K562eGFP-bcr was detected by MTT The apoptosis ofK562control and K562eGFP-bcrWas checked by flow cytometry after Annex-V/PI double staining, Heoechst 33258 stain and DNA ladder, respectively.Results The recombinant plasmid gene expression system was verified through enzymatic cutting and DNA sequencing. The recombinant plasmid had a molecular size of 5.5kb and the target gene fragment which included the phosphorylation site of the 354th amino acid serine had a molecular size of 843bp. Proved by Western-blotting, the molecular weight of the expression product of Bcr gene is 37KD. The proliferation capability K562eGFP-bcr cells was inhibited at 48 hrs after transfection and decreased significantly 72 hrs later. K562eGFP-bcr cells happened the change in morphous at 48 hrs after cultured in vitro and had a typical character of apoptosis 72 hrs after cultured in vitro.Conclusion We have successfully constructed a PEGFP-Bcr system, which carries the integrated expression of bcr gene and green fluorescent protein gene in mammal cells. Th... |
